HUMIC AND FULVIC ACIDS ALLEVIATE OXIDATIVE DAMAGE INDUCED BY HYDROGEN PEROXIDE. Journal

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HUMIC AND FULVIC ACIDS
ALLEVIATE OXIDATIVE DAMAGE
INDUCED BY HYDROGEN
PEROXIDE.
Journal Abd El-Moneim M.R. Afify1*, Osama A.
Konswa1*, Afifi, M.M.I.**
J. Biol. Chem.
Environ. Sci., 2013,
Vol. 8(4): 555-580
www.acepsag.org
*Department of Biochemistry, Faculty of Agriculture, Cairo
University, p.12613, Gamma st., Giza, Cairo-Egypt
**Soils, Water and Environ. Res. Inst., Agric. Res. Center
(ARC), Giza, Egypt.
ABSTRACT
Fulvic acid (FA) and humic acid (HA) are formed through the
degradation of organic substances by chemical and biological process.
FA and HA consists of a mixture of closely related complex aromatic
polymers with the presence of aromatic rings, phenolic hydroxyl,
ketone carbonyl, quinone carbonyl, carboxyl and alkoxyl groups. In
this study, the isolated Humic and fulvic acids of prepared compost
showed that, the values of the phenolic-OH groups were higher than
those of the COOH groups in both of humic and fulvic acids.
Carboxylic and phenolic-OH groups in fulvic acid was higher than
humic acid. In addition, the in vitro antioxidant activity was evaluated
by various antioxidant assays, including superoxide anion radical
scavenging and hydrogen peroxide scavenging activities. All
treatments showed a strong antioxidant activity in tested methods.
Fulvic acids has been found to possess the highest activity in all tested
models in dose dependent manner. Further more in this study, we
examined the safety administration and evaluate the antioxidant
scavenging activity of fulvic and humic acids in comparison with
reference compounds when the tested compounds fulvic acid reflected
the safe usage of these compounds at the recommended doses against
the control rats. On the other hand, treatments of humic acid at
different concentrations with 10,50 100 and 150 mg/ Kg body weight
(B.W.) showed a significant differences in kidney function Urea ,
Creatinine and enzyme activities of the liver function as monitored
via AST and ALT enzyme activities when using 150 mg/ Kg body
weight. (46.7, 2.1, 41.4 and 31) respectively compared to negative

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556
control. Also, the results exhibited a significant concentration-
dependent manner of free radical scavenging activity as well as
significant reduction of MDA level when, the lowest lipid
peroxidation has been reported with 150 mg/kg B.W. fulvic acid. On
the other hand, both of the humic acid and fulvic acid showed a
significant decrease in the enzymes activity of glutathione peroxidase
(GPx) and super oxide dismutase (SOD) indicating their direct
protective effects against induced oxidative stress, via GPx and SOD
defense enzymes modulation. Fulvic acid exhibited more potent
protective activity at a dose of 150 mg/Kg. The antioxidant properties
of the FA and HA partially support the health beneficial properties of
these compounds and therefore, the FA is a good candidate to be used
in pharmaceutical or food industries as an accessible source of natural
antioxidants against oxidative stress induced by accumulated
hydrogen peroxide. The possible application of compost-derived FA
and HA as an antioxidant and human nutritional supplement has been
supported.
Key words: compost characteristics, Fulvic acid, Humic acids, In
vitro and In vivo antioxidant scavenging capacity.
INTRODUCTION
Oxidative stress, induced by oxygen radicals, is believed to be a
primary factor in various degenerative diseases, such as cancer
Muramatsu et al., (1995), atherosclerosis Das, Bandyopadhyay et
al., (1997). Many antioxidant compounds, naturally occurring from
plant sources, have been identified as a free radical or active oxygen
scavengers Zheng and Wang, (2001). Recently, interest has increased
considerably in finding naturally occurring antioxidants for use in
foods or medicinal materials to replace synthetic antioxidants, which
are being restricted due to their side effects such as carcinogenicity Ito
et al., (1983). In addition, natural antioxidants have the capacity to
improve food quality and stability and can also act as nutraceuticals to
terminate free radical chain reactions in biological systems, and thus
may provide additional health benefits to consumers. Increasing
experimental evidence has suggested that these compounds can affect
a wide range of cell biological functions by virtue of their radical
scavenging properties Lai et al., (2001).

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Humic substances (HS) are the most widely-spread natural
complexing ligands occurring in nature. The presence of HS in soils
have also been detected, even in the Antarctic continent where the
humification process under Antarctic conditions is very specific and
different from the other continents Gajdo.ová et al., 2001, Pacheco
and Havel, 2002 and Gajdo.ová et al., (2003).
Humic substances produced on a commercial scale are used in
veterinary and human medicine. Several studies of the medicinal
properties of humic materials have been reported Mund-Hoym,
(1981) and Brzozowski et al., (1994). It was found that humic acids
administered prophylactically to rats decreased significantly the
extension of gastric damage induced by ethanol. Also, significantly
accelerated the ulcers healing process Brzozowski et al., (1994).
Pflug and Ziechman (1982) reported that humic acids are able to
interact with the bacterium Micrococcus luteus.
The possibility of soil humus extract with amino acid complexes
and vitamin B analogues being a candidate as a base of cosmetic and
pharmaceutical products has been studied. The main reason for the
increasing attention devoted to humic acids can be explain by their
antiviral, anti-inflammatory and estrogenic activities Yamada et al.,
(1998). The potential of humic substances to form chelate complexes
with heavy metals (such as cadmium) enable them to be used for the
elimination of heavy metals from living organisms Klöcking, (1992).
On the other hand, humic acid has been shown to be a toxic factor for
many mammalian cells, but the specific mechanism of its cytotoxicity
remains unclear.
Its redox properties make humic acid capable of reducing
iron(III) to iron(II) in aqueous conditions over a broad range of pH
values (from 4.0 to 9.0) and of reducing and releasing iron from
ferritin, but this process is partially inhibited by superoxide
scavengers.This may be one of the most important mechanisms for
HA-induced cytotoxicity Ho et al., (2003). Now it is time for new
applications of humic substances in less traditional arenas, mainly in
biomedicine Laub, 1999; Laub, (2003) and Ghosal, (2003).
Humic acid is known to exhibit electron-transfer reactions within
itself or between HA and other substances, and it also seems to exhibit
some redox capability. The quinone moieties of (HA) may be the
electron-accepting groups, with the resultant hydroquinones donating
electrons to an ultimate electron acceptor such as iron (III), such that

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558
the ability of (HA) to participate in such an electron transfer system
may thus be based on its quinone/ hydroquinone redox couple Lovely
et al., (1996).
Fulvic acid has various useful effects due to its functional
groups. Studies on the physiological actions of (FA) exerted on the
living body are gradually being carried out. The possible application
of coal-derived FA as an antimicrobial and antioxidant substance has
been described and the inflammatory property of coal-derived (FA)
has been also reported Van Rensburg et al., (2000) and Ueda et al.,
(2004). It has been used externally to treat haematoma, phlebitis,
desmorrhexis, myogelosis, arthrosis, polyarthritis, osteoarthritis and
osteochondrosis. Likewise, (FA) has been taken orally as a therapy for
gastritis, diarrhoea, stomach ulcers, dysentery, colitis and diabetes
mellitus Schepetkin et al., (2009). (FA) and (HA) substances isolated
from soil and water reservoirs have been reported to stimulate
neutrophil and lymphocyte immune function Schepetkin et al.,
(2003). (FA) and its related compounds have no toxic compounds
Bergh et al., (1997). (HA) and FA are commonly used as a soil
supplement in agriculture and as a human nutritional supplement.
ROS contribute to the development of various diseases such as
atherosclerosis, diabetes, cancer, neurodegenerative diseases, liver
cirrhosis and ageing process Basaga, (1990). To prevent the damage
caused by reactive oxygen species (ROS), tissues had developed an
antioxidant defense system that includes nonezymatic antioxidants
(e.g., glutathione, uric acid, bilirubin and vitamins C and E) and
enzymatic activities such as superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GPx) Sorg, (2004). A second level
of prevention against ROS- induced damage is constituted by
scavenging compounds, which are able to reduce the incidence of free
radical-mediated diseases Sorg, (2004).The use of antioxidants, both
natural and synthetic, in the prevention and cure of various diseases is
expanding. There is a considerable interest in the antioxidant activities
of molecules such as HA, FA and plant polyphenolic components Jin
et al., (2005). In this sense, FA displays activity against superoxide
and hydroxyl radicals Wang et al., (1996). Despite the broad
spectrum use of FA for a variety of medical conditions, little is known
regarding the mechanisms of action of FA. Therefore, the objective of
this work was to evaluate, the in vitro superoxide (O2
•–), hydrogen

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559
peroxide (H2O2), scavenging capacity of the HA and FA derived from
a compost against induced oxidative damage induced by administrated
hydrogen peroxide.
MATERIALS AND METHODS
Characteristics of compost samples
Compost and biogas manure analysis
The sample analyzed for its physical, chemical and biological
characteristics.
I. Physical analyses
a. Density
Density was determined using the core method according to
Vomocil (1965).
b. Moisture content
Compost samples were dried at 70 ºC to constant weights Page
et al., (1982). The moisture content was calculated as a percentage of
fresh weight.
C. pH value
The pH values of compost were determined in 1:10 compost-
water extract using a glass electrode of Orion Expandable ion analyzer
EA920 according to Jodice et al. (1982).
d. Electrical conductivity (EC)
Electrical conductivity measurements were run in (1:10)
compost: water extract Richards, (1954), using EC meter ICM model
71150.
II. Chemical analyses
a. Organic matter (OM)
Organic matter content of compost material was determined as
recommended by Page et al. (1982).
b. Organic carbon (OC)
Organic carbon content was calculated by multiplying the dry
organic matter by 58 %, Jackson, (1973).
c. Total nitrogen (TN)
Total nitrogen was determined in compost using Kjeldahl
digestion method reported by Jackson (1973).

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d. Soluble nitrogen (ammonium and nitrate-nitrogen)
Soluble nitrogen forms in compost i.e. NH4+ and NO3- were
determined according to the methods outlined by Page et al., (1982).
e. Total phosphorus (TP)
Acid solution of the digested compost was used for total
phosphorus content using ascorbic acid as a reluctant Murphy and
Riley, (1962) as modified by John (1970) and outlined by Page et al.,
(1982). A Perkin Elmer spectrophotometrically using ammonium
molybdate and ascorbic acid reagents.
f. Total potassium (TK)
Digest solutions of compost and biogas manure were subjected
for determination of total potassium content flame photo metrically
Chapman and Pratt, (1961).
III. Biological analyses
a. Determination of total counts of bacteria, fungi and
actinomycetes
The number of total viable bacteria, fungi and actinomycetes
was determined as reported by Reinhold et al., (1985) and Allen,
(1950).
b. Coliform group bacteria (total and faecal) and Salmonella
and Shigella
Samples were assayed as described by Difco. Manual (1977).
2. Extraction and purification of humic and fulvic acids
I. Extraction
Extraction of humic substances was run according to the
methods described by Sanchez-Monedero et al., (2002).
II. Humic acid Purification
The humic acid was purified according Chen et al., (1978).
III. Fulvic acid purification
The purification method of fulvic acid was run according to
Kononova (1966) and Susilawati et al., (2007).
IV. Characteristics of of humic and fulvic acids extracted from compost.
a. Determination of total acidity
Total acidity of humic and fulvic acids was determined
following the method described by Dragunova (1958).

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b. Determination of Carboxyl group
Carboxyl group of humic and fulvic acids was determined by
calcium acetate Ca (CH3COO)2 methods mentioned by Schnitzer and
Gupta (1965).
c. Elemental analysis(C, H, N, S and O2 %)
Elemental analysis for carbon, hydrogen, nitrogen and sulphur
contents of the purified humic and fulvic acids were performed by gas
chromatography on a Hewlett-Packard 185 (C, H, N, S automatic)
microanalysis (Vario Elmentor / C, H, N, S Germany) (Micro
Analytical center, Faculty of Science, Cairo University). Oxygen was
calculated by subtracting total amount of carbon%, hydrogen %,
nitrogen % and sulphuer % from 100 Goh and Stevenson (1971). All
results were calculated as percentage.
3. Hydrogen peroxide and superoxide radical Scavenging Activity
3.1. Assay of superoxide radical scavenging activity
The assay was based on the capacity of the humic and fulvic
acids to inhibit formazan formation by scavenging the superoxide
radicals generated in riboflavin–light–Nitro Blue Tetrazolium system
Beauchamp and Fridovich, (1971). Each 3ml reaction mixture
contained 50mM sodium phosphate buffer (pH 7.6), 20 mg riboflavin,
and 12mM EDTA, and 0.1 mg NBT and 1ml sample solution.
Reaction was started by illuminating the reaction mixture with
different concentrations of sample extract (10, 50,100 amd 150 µg/ml)
for 90 s. Immediately after illumination, the absorbance was measured
at 590 nm. The entire reaction assembly was enclosed in a box lined
with aluminium foil. Identical tubes with reaction mixture were kept
in the dark and served as blanks. The percentage inhibition of
superoxide anion generation was calculated using the following
formula:
Where A0 is the absorbance of the control, and A1 is the
absorbance of the samples/standard.
3.2. Determination of H2 O2scavenging capacity
The ability of humic and fulvic acids extracts to scavenge
hydrogen peroxide was determined according to the method of Ruch

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562
et al (1989). A solution of hydrogen peroxide (2 mmol/l) was
prepared in phosphate buffer (pH 7.4). Hydrogen peroxide
concentration was determined spectrophotometrically from absorption
at 230nm . Humic and fulvic acids extracts (10–150 mg/ml) were
added to hydrogen peroxide solution (0.6 ml). Absorbance of
hydrogen peroxide at 230 nm was determined after 10 min against a
blank solution containing phosphate buffer without hydrogen
peroxide. For each concentration, a separate blank sample was used
for background subtraction. The percentage inhibition activity was
calculated from [(A0_A1)/A0] x100, where A0 is the absorbance of the
control and A1 is the absorbance of the samples/standard.
4. Pharmacological and biological activity of compounds humic
and fulvic acids.
Experimental animals
Rats used in this study were procured from The Animal House
Colony at the National Research Centre (NRC), Egypt. Wistar rats
were utilized. All animals were housed under standard conditions of
natural 12 h light and dark cycle with free access to food and water.
Animals were allowed to adapt to the laboratory environment for one
week before experimentation. All animal procedures were performed
after approval from the Ethics Committee of The National Research
Centre- Egypt and in accordance with the recommendations of the
proper care and use of laboratory animals.
4.1. In vivo safety administration of humic acid and vulvic acid
Biological evaluation( Kidney and liver function)
Experimental design
Male rats (125-175 g) were divided into 9 groups (eight rats/
group). The first group represented the negative control, was
administered 5% dimethyl sulphoxide (DMSO. Groups two, three,
four and five were treated with different concentrations of humic acid
(10, 50 ,100 and 150 mg/Kg body weight). Groups six , seven, eight
and nine were treated with analogous concentrations of fulvic acid.
After four weeks rats were decapitated, samples were taken from
various groups and prepared according to each method.
4.1.1. Blood urea
It was estimated by the enzymatic method as described by
Patton and Couch (1977).

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4.1.2. Serum creatinine
It was determined according to the method described by
Faulkner and King (1976).
4.1.3. The activity of aspartate transaminase (AST) and alanine
transaminase (ALT)
It was estimated according to the method descibed by (Reitman
et al., 1957).
4.2.In vivo estimation of antioxidant and biological function.
In vivo estimation of antioxidant enzymes activity
Experimental design
Sprague - Dawley male rats (125-175 g) were divided into 11
groups (eight rats/ group). The first group represented the negative
control, was administered 5% dimethylsulphoxide (DMSO). The
second group, the positive control, was administered an oral dose of
H2O2 + 5 % DMSO to induce oxidative damage. The single dose/week
was 2 mL/Kg body weight. Groups three, four, five and six were
treated with different concentrations of humic acid (10, 50, 100 and
150 mg/ Kg body weight). Groups seven, eight, nine and ten were
treated with analogous concentrations of fulvic acid . Group eleven
received an oral dose of Ascorbic acid 10 mg/ Kg body weight + 5%
DMSO. After four weeks rats were decapitated, samples were taken
from various groups and prepared according to each method.
4.2.1. Assay of Glutathione peroxidase (GPx) activity
Glutathione peroxidase was estimated by the method of
(Pagila and Valentine 1967).
4.2.2. Assay of superoxide dismutase (SOD) activity
The activity of SOD was assayed by measuring its ability to
inhibit the photochemical reduction of NBT using the method of
Beauchamp and Fridovich (1971). The 3 ml reaction mixture
contained 50 mM phosphate buffer pH 7.8, 13 mM methionine, 75
µM NBT, 2 µM riboflavin, 1.0 mM EDTA and 20 µl enzyme extract.
Riboflavin was added last and the reaction was initiated by placing the
tubes 30 cm below 15 W fluorescent lamps. The reaction was started
by switching on the light and was allowed to run for 10 min.
Switching off the light stopped the reaction and the tubes were
covered with a black cloth. Non-illuminated tubes served as control.
The absorbance at 560 nm was read. The volume of enzyme extract

HUMIC AND FULVIC ACIDS ALLEVIATE OXIDATIVE DAMAGE
564
corresponding to 50% inhibition of the reaction was considered as one
enzyme unit.
4.2.3. Estimation of lipid peroxidation and nitric oxide Lipid
peroxidation assay (malonaldehyde (MDA) contents)
Fresh liver samples (200 mg) were homogenized in 2 ml of 0.1%
(w/v) trichloroacetic acid (TCA), followed by centrifugation at
12,000×g for 20 min. The supernatant (1 ml) obtained was mixed with
an equal volume of TCA (10%) containing 0.5% (w/v) Thiobarbituric
acid (TBA) (or no TBA as the blank), and heated at 95°C for 30 min,
then cooled in ice. The reaction product was centrifuged at 12,000×g
for 15 min and the supernatant absorbance was measured at 400, 532
and 600 nm. The MDA equivalent was derived from the absorbance
according to) Hodges et al., (1999).
RESULTS AND DISCUSSION
Composting of organic wastes is a biooxidative process
involving the mineralization and partial humification of the organic
matter, leading to an establishment of final product, free of
phytotoxicity and pathogens with certain humic properties Zucconi
and de Bertoldi, (1987).
The factors affecting the composting for production of high
quality compost with added agricultural value, focusing on the organic
matter (OM) humification, nutrient content, maturity degree and testes
of safety Moral et al., (2009).
1. Characteristics of compost sample.
Data presented in Table (1) showed that the density of the
composting materials for compost used for extraction of humic
substances was 629 kg/m3, to produce (not exceed to 700 Kg / m3) for
good compost material. Ammonia contents in compost materials is 35
mg/ Kg and these concentration was in the range of recommended
levels for mature compost material Zucconi and de Bertoldi, (1987).
As shown in Table (1) the organic matter in compost sample
exceeded 20% as a minimum limit stated by Ge et al., (2006) for
mature compost.
The C: N ratio in sample was 13:1 resembling the optimum less
than 25: 1 for the mature compost as stated by TMECC (2002).
It is worthy to notice that most of the compost sample recorded
no existence of pathogenic bacteria, parasites, seed weeds or

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nematodes and these results were safe limits for composts stated by
Iannotti et al., (1993).
Table (1): Physical, chemical and biological analyses of compost
materials.
2. Characteristics of of humic and fulvic acids extracted from
compost.
Results in Table (2) showed that the total acidity of the tested
humic acid sample recorded 165 m mol /100g acid , while the total
acidity in fulvic acid 500 m mol / 100 g . The values of -COOH and
phenolic-OH groups showed that the values of the phenolic-OH
groups were higher than those of the -COOH groups in both of humic
and fulvic acids. Carboxylic and phenolic-OH groups in fulvic acid
was higher than humic acid.

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566
Elementary analysis of humic and fulvic acids on their content of
C, N, H, O2 and S. humic acid content from C, N, H, and S higher
than fulvic acid . On the other hand fulvic acid content of O2 grater
than humic acid contents.
Table (2): Major characteristic of humic and fulvic acids
extracted from compost.
3. In vitro antioxidant activity of humic and vulvic acids.
3.1 .Hydrogen peroxide and superoxide radical Scavenging
Activity
Superoxide radical is known to be a very harmful species to
cellular components as a precursor of more reactive oxygen species
Halliwell (1994). The superoxide radical is known to be produced in
vivo and can result in the formation of H2O2 via dismutation reaction.
Moreover, the conversion of superoxide and H2O2 into more reactive
species, e.g., the hydroxyl radical, has been thought to be one of the
unfavourable effects caused by superoxide radicals Halliwell,
(2001).The humic acid and fulvic acid treatments are found to be an
efficient scavenger of superoxide radical generated in riboflavin–
NBT–light system in vitro and their activity are in comparable to that
of ascorbic acid. Photochemical reduction of flavins generates O2,
which reduces NBT, resulting in the formation of blue formazan
Beauchamp and Fridovich, (1971). From this experiment, it is noted
that the inhibition of the formation of blue formazan and also the
percentage inhibition are directly proportional to the concentration of
the treated humic acid and fulvic acid shown in table (3) and
illustrated in Fig.(1) reported that, the percentage of superoxide
radical scavenging is determined with 10,50,100 and 150 (µg/ml) of
the of humic acid and fulvic acid represented the highest Scavenging
Activity expressed as % inhibition is as follows: humic acid (67.12%)
and vulvic acid (79.00 %) against ascorbic acid standard at the same

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567
concentration (100 %). Scavenging activities on hydrogen peroxide.
This result clearly indicates that the tested treatments have a
noticeable effect on scavenging superoxide radical in an amount-
dependent manner. These data was in accordance with Cos et al.,
(1998) who reported that, phenolic hydroxyl group is the main active
group which scavenges OH• (this effect can be primarily attributed to
the hydrogen donation and electron transfer capacities of OH group)
and favours the encapsulation of the pro- oxidant iron species, which
generates OH• through the fenton reaction Butkovic et al., (2004).
This suggests that the phenolic hydroxyl group and metal-chelating
ability by FA could explain the ROS scavenging activity observed.
Table (3): Superoxide Radical Scavenging Activity expressed as %
inhibition.
Fig. (1): Humic and vulvic acid represent superoxide radical
Scavenging Activity expressed as % inhibition.
3.2. Hydrogen peroxide (H2O2) scavenging activity
Humic acid and Vulvic acid are classes of compounds consisting
of complex polymeric aromatic structures. It is formed through

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568
environmental degradation of animal, plant, fungal and bacterial
biopolymersChoudhry, (1984) and Chen (1996). The ability of
humic acid and fulvic acid to scavenge hydrogen peroxide is
determined according to the method of Ruch et al., (1989). The
scavenging ability of various treatments with hydrogen peroxide is
shown in table (4) and illustrated in Fig. (2) compared with the
ascorbic acid as standards. It is noticed that all treatments are capable
of scavenging hydrogen peroxide in an amount-dependent manner.
The percentage of scavenging hydrogen peroxide is determined with
150 (µg/ml) of the of humic acid and fulvic acid represented the
highest scavenging activity and expressed as % inhibition as follows:
humic acid (56.70%) and vulvic acid (67.22 %) against ascorbic acid
standard at the same concentration (91.54%). Dealing with the
scavenging activities on hydrogen peroxide, the results reveal that all
the treatments have the scavenging character in accordance with the
standards.
Table (4): Hydrogen peroxide Scavenging Activity expressed as
% inhibition.
Fig. (2): Humic and fulvic acid Scavenge hydrogen peroxide
expressed as % inhibition

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569
4. Biological evaluation of humic acid and vulvic acid
4.1. In vivo safety administration of humic acid and vulvic acid.
In vivo assay of kidney and liver functions.
The obtained data in Table (5) revealed that, humic acid and fulvic
acid were tested for their possible effect on kidney and liver functions to
assess their safety in vivo investigation. The results cleared that urea and
creatinine levels as kidney function did not shows any significant
toxicity when using fulvic acid with concentration of 10,50 ,100 and 150
mg/ Kg compared to negative control. On the other hand treatments of
humic acid of different concentration with 10,50 and 100 mg/ Kg show
significantly differences in kidney function Urea (mg/dl) , Creatinine (
mg/dl) and AST and ALT (U/ml) enzyme activities of the liver by using
150 mg/ Kg b.w. (46.7 , 2.1 , 41.4 and 31 ) respectively compared to
negative control . These data was in accordance with Kuo-Jang et al.,
(2003) who reported that, low concentrations of Humic Acid , such as 25
and 50 µg/ml, reveal no increase in Humic Acid induced lipid
peroxidation compared that occurring under control conditions. Also, the
data reflected that the high doses of humic acid significantly affected the
liver function and the kidney function that is may be due to the toxicity
dose dependence of humic acid affecting the plasma membrane integrity
and toxicity .Nevertheless, Huang et al., (1995) reported that ,The daily
intake of HA by the average resident in these areas has been estimated to
be as high as 400 mg. It is conceivable that the intake of large quantities
of HA has adversely affected the health of inhabitants in these areas.
Radioisotope tracing with iodinated HA in rats indicated that up to 60%
of HA remained in the body 24 h after its administration . The oxy or
hydroxy functional groups in HA can interact with cell membrane
proteins and HA remaining thus in the body is not easily cleared. On
long-term exposure to HA, it can accumulate and rise to cytotoxic
concentrations locally in cells or in some region of body. In addition,
Previously, Humic Acid disrupts intracellular calcium homeostasis and
HA enhances the permeability of cell membranes to extracellular Ca+2,
resulting in sustained elevation of cytosolic Ca+2 Yang, (1994).
Also These data was in accordance with Noemí et al., (2011), who
found that , The antioxidant properties of fulvic acid explain some of the
health beneficial effects of this compound since the excessive production
of O2•, HOCl, H2O2, OH•, ONOO– and O2 are involved in several
pathologies. Moreover, they could be a good candidate for use in
pharmaceutical or food industries as an accessible source of natural

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570
antioxidants and for the improvement of food quality by retarding lipid
oxidation.
Table (5): Cytotoxicity effect of various concentrations of humic
and fulvic acids on urea, creatinine and AST and ALT in rats
4.2. In vivo estimation of antioxidant defense enzyme activities
affected by administrated humic acid and fulvic acid under
hydrogen peroxide oxidative stress
Hydrogen peroxide-induced damage of liver cells results in
oxidative damage through free-radical generation. In this work, the
rats were treated with a single oral dose of hydrogen peroxide
(2 ml/Kg body weight / week) to induce oxidative stress and liver
damage. Humic acid and fulvic acid were evaluated for their
protective effect against oxidative stress and induced liver damage in
rats by estimating SOD, GPx activity as well as MDA levels. The
results shown in table (6) and illustrated in fig (3) and fig.(4) revealed
that, the administrated humic acid and fulvic acid showed a significant
reduction in both enzymes indicating their protective effects against
induced oxidative damage, via GPx and SOD defense enzymes
modulation. Fulvic acid exhibited more potent protective activity at a
dose of 150 mg/Kg.

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Table (6): The antioxidant activity of compounds fulvic acids and
humic substances against MDA formation, SOD and GPX in control
and treated rats with H2O2 oxidative stress.
The data also revealed a significant decrease in MDA level upon
treatment with different concentrations of humic acid and fulvic acid.
The best result was shown by Fulvic acid at a dose of 150 mg/Kg,
which may be attributed to its higher antioxidant activity compared to
humic acid. The data represented that, the humic acid and vulvic acid
exhibited asignificant concentration-dependent free radical scavenging
activity as well as significant reduction of MDA level. Test
compounds humic acid and fulvic acid demonstrated the highest free
radical-scavenging activity and the lowest MDA content.
Furthermore, both test compounds, humic acid and fulvic acid,
showed protective effect against lipid peroxidation in hepatic
microsomes. This effect may be due to their inhibitory effect on
inducible nitric oxide synthase (iNOS) and scavenging effect on
peroxynitrite formation. The lowest MDA level was observed with
fulvic acid compound at a dose of 150 mg/Kg bw, (Table (4) and Fig.
(4). This finding is in accordance with previous studies showing that,
the basic structure of the humic acid and fulvic compounds has an

HUMIC AND FULVIC ACIDS ALLEVIATE OXIDATIVE DAMAGE
572
important antioxidant activity. In addition, humic acid and fulvic
compounds were able to prevent glutathione oxidation induced by
H2O2.
Fig. (3): The modulating effect of administrated fulvic acids and
humic substances on SOD activity under oxidative
stress.
Fig. (4): The modulating effect of administrated fulvic acids and
humic substances on GPX activity and MDA formation
under oxidative stress.
The results reflected the safe usage of Fulvic acids at the
recommended doses against the control rats. In vivo examination of
the active compounds humic acid and fulvic acid exhibited a good
activity against lipid peroxidation and free radical scavenging in a

J. Biol. Chem. Environ. Sci., 2013, 8(4), 555-580
573
dose of 150 mg/Kg bw. In addition, both compounds demonstrated
high protective effect against H2O2-induced liver damage. In
accordance ,The FA act as an antioxidant like other high molecular
weight plant phenolics such as tannins . In this present study, FA in
vitro scavenged of O2•– and H2O2, in a concentration-dependent way
Ueda, 2004. These specific scavenging properties of humic acid and
fulvic acid contribute to explain their antioxidant properties
.Therefore, the antioxidant effect of compounds humic acid and fulvic
against lipid peroxidation seems to be interesting for the treatment of
neurodegenerative disorders. FA also reduced OH• radical formation,
rate and time dependent, in aqueous iron-hydrogen peroxide reaction
Lindsey and Tarr (2000). The hydrophobicity of the antioxidants
plays a role in the efficacy of inhibition. Baigorri, et al., ( 2008). And
Baigorri, et al., (2009) found that, the presence of structural units O-
functionalized, including aromatic domains in FA, could explain their
tendency to form molecular aggregates (hydrogen bridges, metal
bridges and hydrophobic interactions). Other studies are necessary to
evaluate the therapeutic potential of these compounds in models of
neurodegenerative disorders in vivo.
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ﻦﻴﺟورﺪﻴﻬﻟا ﺪﻴﺴآا قﻮﻔﺑ ﺔﻠﻣﺎﻌﻤﻟا ﻦﻋ ﻢﺟﺎﻨﻟا ىﺪﺴآﺄﺘﻟا رﺮﻀﻟا ﻒﻴﻔﺨﺗ
ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺣو ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺣ ﺔﻄﺳاﻮﺑ
ا.د .ﻰﻔﻴﻔﻋ ﺪﻤﺤﻣ ﻢﻌﻨﻤﻟا ﺪﺒﻋ*د .ﺪﻤﺣا ةﻮﺼﻨﻗ ﺔﻣﺎﺳا*د .ﻰﻔﻴﻔﻋ ﻢﻴهاﺮﺑا دﻮﻤﺤﻣ ﺪﻤﺤﻣ**
*ﺴﻗ ﻢﺔﻳﻮﻴﺤﻟا ءﺎﻴﻤﻴﻜﻟا - ﺔﻋارﺰﻟا ﺔﻴﻠآ –ةﺮهﺎﻘﻟا ﺔﻌﻣﺎﺟ
** ﺎﻴﺟﻮﻟﻮﻴﺑوﺮﻜﻴﻤﻟا ﻢﺴﻗ–ﻣ ﺔﺌﻴﺒﻟاو ةﺎﻴﻤﻟاو ﻰﺿارﻻا ثﻮﺤﺑ ﺪﻬﻌ-ﺔﻴﻋارﺰﻟا ثﻮﺤﺒﻟا ﺰآﺮﻣ-
ﺮﺼﻣ ةﺰﻴﺠﻟا
ﻩﺪﺴآﻻا تﺎﻴﻠﻤﻋ ﻦﻣ ﺔﻋﻮﻤﺠﻣ لﻼﺧ ﻦﻣ ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺣو ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺣ نﻮﻜﺘﻳ
ﻦﻣ ﺪﻳﺪﻌﻟا دﻮﺟﻮﺑ تﺎﺒآﺮﻤﻟا ﻩﺬه ﺰﻴﻤﺘﺗ ﺚﻴﺣ ﺔﻴﺟﻮﻟﻮﻴﺒﻟا تﺎﻴﻠﻤﻌﻟا ﻦﻣ ﺔﻋﻮﻤﺠﻣ لﻼﺧ ﺔﻳﻮﻴﺤﻟا
ﻟا تﺎﻋﻮﻤﺠﻣو تﻻﻮﻨﻴﻔﻟاو ﺔﻳﺮﻄﻌﻟا تﺎﻘﻠﺤﻟاﺪﻗو ﻞﻴﺴآﻮﺑﺮﻜﻟا تﺎﻋﻮﻤﺠﻣو نﻮﻨﻴﻜﻟاو نﻮﺘﻴﻜ
ﺾﻣﺎﺣ ﻦﻣ ﻞآ ﻰﻓ ﻪﻌﻔﺗﺮﻣ ﺔﻴﻟﻮﻨﻴﻔﻟا تﺎﻋﻮﻤﺠﻤﻟا ﻢﻴﻗ نا ﺞﺋﺎﺘﻨﻟا تﺮﻬﻇا ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺣو
ﻚﻴﻔﻟﺎﻔﻟا .ﻢﺗ ، ﻚﻟذ ﻰﻟإ ﺔﻓﺎﺿﻹﺎﺑو ﻢﻴﻴﻘﺗ طﺎﺸﻨﻟا ﺎﻬﻨﻣو ﺔﻔﻠﺘﺨﻣ قﺮﻃ ﻞﺒﻗ ﻦﻣ ةﺪﺴآﻸﻟ دﺎﻀﻤﻟا
ﻮﻓ ﻚﻟﺬآو نﻮﻴﻧا ﺪﺴآا ﺮﺑﻮﺴﻠﻟ ةﺮﺤﻟا قﻮﻘﺸﻠﻟ تادﺎﻀﻤﻟا ﻊﻴﻤﺟ ﺮﻬﻇأ و ﻦﻴﺟورﺪﻴﻬﻟا ﺪﻴﺴآا ق

HUMIC AND FULVIC ACIDS ALLEVIATE OXIDATIVE DAMAGE
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ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺣ ةرﺪﻗ نا ﺪﺟوو ﺎهرﺎﺒﺘﺧا قﺮﻃ ﻲﻓ يﻮﻗ ةﺪﺴآﻸﻟ دﺎﻀﻣ طﺎﺸﻧ تﻼﻣﺎﻌﻤﻟا
ماﺪﺨﺘﺳا نﺎﻣا ﺞﺋﺎﺘﻨﻟا تﺪآاو ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺣ تﺎﺒآﺮﻣ طﺎﺸﻧ ﻦﻣ ﺮﺒآا ةﺪﺴآﻼﻟ دﺎﻀﻤآ
ﻦﻣ ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺤﺑ ﺔﻧرﺎﻘﻣ ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺣ تﺎﺒآﺮﻣ ﺪﺒﻜﻟا تﺎﻤﻳﺰﻧا طﺎﺸﻧ تاﺮﺷﺆﻣ لﻼﺧ
ﻦﻴﻨﻴﺗﺎﻳﺮﻜﻟاو ﺎﻳرﻮﻴﻟا ﺮﻳﺪﻘﺘﺑ ﻰﻠﻜﻟا طﺎﺸﻧ ﻚﻟاﺬآو.
ﺾﻣﺎﺣ ﻦﻣ ﺮﺒآا ﻪﺟرﺪﺑ ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺤﻟ ةﺪﺴآﻼﻟ دﺎﻀﻣ ﺮﻴﺛﺎﺗ دﻮﺟو ﺞﺋﺎﺘﻨﻟا تﺮﻬﻇا ﺎﻤآ
ﺖﺴﻜﻋ ﺪﻗ ﺔﻴﻤﺴﻟا ﺞﺋﺎﺘﻧ ﻦﻜﻟو ﻚﻴﻣﻮﻴﻬﻟاﺰﻴآﺮﺘﻟا ﺔﻧرﺎﻘﻣ ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺣ تﻼﻣﺎﻌﻤﻟ ﻦﻣﻻا
ﻚﻴﻣﻮﻴﻬﻟا ﺾﻣﺎﺣ تاﺰﻴآﺮﺘﺑ ﺰﻴآﺮﺘﻟا نﺎآ ىﺮﺧا ﺔﻬﺟ ﻦﻣ ،150 ﻦﻣ ماﺮﺟ ﻮﻠﻴآ ﻞﻜﻟ ماﺮﺠﻠﻠﻣ
ﻰﻓ ﻼﺜﻤﺘﻣ ﻦﻴﺟورﺪﻴﻬﻟا ﺪﻴﺴآا قﻮﻔﺑ ةﺪﺴآﻼﻟ ﺔﻣﺎﺴﻟا تاﺮﻴﺛﺎﺘﻠﻟ دﺎﻀﻤﻟا ﻩﺮﻴﺛﺎﺗ ناﺮﺌﻔﻟا نازوا
ﺎﻤﻣ ﺔﻳﻮﻴﺤﻟا ﺔﺠﺴﻧﻻا ﻰﻓ تاﺪﻴﺒﻴﻠﻟا ةﺪﺴآا ﻦﻣ نﻮﻜﺘﻤﻟا ﺪﻴهﺪﻟﺎﻧﻮﻟﺎﻤﻟا ﻢآاﺮﺗ تﻻﺪﻌﻣ ﻰﻓ ضﺎﻔﺨﻧا
ماﺪﺨﺘﺳﻻ ﻰﺋﺎﻗﻮﻟا ﺮﻴﺛﺎﺘﻟا ﺲﻜﻌﻳ ﻪﺘﻓﺎﺿا ﻦﻜﻤﻳو ةﺪﺴآﻼﻟ دﺎﻀﻤآ ﻚﻴﻔﻟﺎﻔﻟا ﺾﻣﺎﺤﻟ ﻦﻣا
طﺎﺸﻧ ﻰﻓ ظﻮﺤﻠﻣ ضﺎﻔﺨﻧا لﻼﺧ ﻦﻣ ﺮﻬﻇ ﺎﻤآ ﺔﻴﻌﻴﺒﻃ ةﺪﺴآﻼﻟ ةدﺎﻀﻣ داﻮﻤآﺔﻴﺋاﺬﻏ تﻼﻤﻜﻤآ
تﺎﺒآﺮﻤﻟا ﻩﺬﻬﻟ ةﺪﺴآﻼﻟ دﺎﻀﻤﻟا ﺮﻴﺛﺎﺘﻠﻟ ةﺮﺷﺎﺒﻣ ﺔﺠﻴﺘﻨآ ةﺪﺴآﻼﻟ ةدﺎﻀﻤﻟا تﺎﻤﻳﺰﻧﻻا.